Lactobacillus helveticus BT-B60 with the function of improving osteoporosis and application thereof

Abstract

The application discloses a Lactobacillus helveticus BT-B60 with the function of improving osteoporosis and application thereof, the Lactobacillus helveticus BT-B60 (Lactobacillus helveticus BT-B60) is preserved in the China Center for Type Culture Collection, the preservation date is November 20, 2025, and the preservation number is CCTCC NO:M 20252624.Compared with the prior art, the application provides a brand-new Lactobacillus helveticus BT-B60, and experimental results show that the strain can promote osteoblast proliferation, inhibit osteoclast formation, improve bone density and bone strength, and improve osteoporosis.The strain has a clear antioxidation protective effect on liver cells, effectively inhibits liver oxidative stress damage, and the extracellular vesicles prepared from the strain BT-B60 have excellent skin antioxidation function, embodying the innovative characteristics of multi-target and multi-tissue antioxidation of the strain.In addition, the strain can also inhibit melanin synthesis, and has multiple effects in whitening in cooperation with the local antioxidation effect of skin.

Description

Technical Field

[0001] This invention belongs to the field of Lactobacillus helveticus technology, and particularly relates to a strain of Lactobacillus helveticus BT-B60 that has the function of improving osteoporosis and its application. Background Technology

[0002] Osteoporosis is a systemic metabolic bone disease characterized by bone loss and deterioration of bone microstructure, directly resulting in increased bone fragility and a significantly elevated risk of fractures. With the accelerating aging of the global population, osteoporosis has become a serious public health problem threatening the quality of life of middle-aged and elderly people. Statistics show that approximately 200 million people worldwide suffer from osteoporosis, especially postmenopausal women. Due to the sharp drop in estrogen levels leading to relatively increased osteoclast activity, bone resorption far exceeds bone formation, resulting in a dramatic increase in the incidence of osteoporotic fractures (such as hip and vertebral fractures).

[0003] Currently, clinical intervention strategies for osteoporosis mainly fall into two categories: nutritional supplements (calcium, vitamin D, etc.) and drug therapy (bisphosphonates, selective estrogen receptor modulators, denosumab, etc.). However, long-term use of these drugs has significant limitations: some patients have low absorption and utilization efficiency of nutritional supplements; and long-term use of bisphosphonates may increase the risk of serious adverse reactions such as atypical femoral fractures and osteonecrosis of the mandible. Therefore, developing safe, effective, and long-term suitable functional foods or biological agents has become a research hotspot in the field of osteoporosis prevention and treatment.

[0004] Probiotics, as an important means of regulating the gut microbiota, have been proven to have the potential to regulate host physiological functions in various experimental models through mechanisms such as reducing intestinal inflammation levels, upregulating the expression of intestinal tight junction proteins, and producing short-chain fatty acids (SCFAs). Therefore, screening probiotic strains with specific functional properties and developing them into functional foods or biological agents has become a current research hotspot.

[0005] Lactobacillus helveticus is an important member of the lactic acid bacteria family and has long been widely used in the dairy fermentation industry. Studies have shown that Lactobacillus helveticus possesses excellent proteolytic capabilities, releasing a variety of bioactive peptides. For example, research has found that its fermentation products inhibit angiotensin-converting enzyme (ACE) activity and show potential in improving cognitive function. However, although the functional diversity of Lactobacillus helveticus has been partially revealed, the screening, identification, and specific applications of highly efficient Lactobacillus helveticus strains with specific functions in food or pharmaceuticals still require further development. Summary of the Invention

[0006] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a strain of Lactobacillus helveticus BT-B60 with osteoporosis-improving function and its application.

[0007] Technical solution: To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a strain of Lactobacillus helveticus with osteoporosis-improving function, wherein the Lactobacillus helveticus is Lactobacillus helveticus BT-B60, which is deposited at the China Center for Type Culture Collection on November 20, 2025, with accession number CCTCC NO: M 20252624.

[0009] In a second aspect, the present invention provides a composition comprising the aforementioned Lactobacillus helveticus BT-B60.

[0010] As a specific embodiment, the composition is a lyophilized powder containing *Lactobacillus helveticus* BT-B60; preferably, the activity of the strain in the lyophilized powder is 1 × 10⁻⁶. 10 ~1×10 12 CFU / g.

[0011] Thirdly, the present invention provides a fermentation medium for *Lactobacillus helveticus* BT-B60, the fermentation medium comprising the following components:

[0012] Soybean peptone 10-14 g / L, yeast extract 6-10 g / L, hydrolyzed whey protein 4-8 g / L, anhydrous glucose 20-30 g / L, xylooligosaccharides 4-8 g / L, calcium carbonate 1-3 g / L, dipotassium hydrogen phosphate 1-3 g / L, potassium dihydrogen phosphate 0.5-2.5 g / L, sodium acetate 4-8 g / L, magnesium sulfate 0.2-1.0 g / L, manganese sulfate 0.1-1.0 g / L, L-cysteine ​​hydrochloride 0.8-1.4 g / L, Tween 80 0.5-1.5 mL / L, pH adjusted to 6-7.

[0013] Fourthly, the present invention provides the use of the aforementioned Lactobacillus helveticus BT-B60 and the aforementioned composition in the preparation of products that improve osteoporosis.

[0014] Fifthly, the present invention provides the use of the aforementioned Lactobacillus helveticus BT-B60 and the aforementioned composition in the preparation of products that inhibit oxidative damage to the liver.

[0015] In a sixth aspect, the present invention provides the use of the aforementioned Lactobacillus helveticus BT-B60 and the aforementioned composition in the preparation of products that inhibit melanin synthesis.

[0016] In a seventh aspect, the present invention provides an extracellular vesicle prepared using the aforementioned Lactobacillus helveticus BT-B60.

[0017] Eighthly, the present invention provides a method for preparing the aforementioned extracellular vesicles, comprising the following steps:

[0018] (1) Centrifuge the fermentation broth of Lactobacillus helveticus BT-B60 cultured to the stable growth phase to obtain metabolic supernatant and Lactobacillus helveticus BT-B60 cells. Filter the metabolic supernatant to further remove cell debris.

[0019] (2) Add exosome extraction reagent to the filtrate obtained in step (1), mix well, let stand, then centrifuge to precipitate extracellular vesicles, resuspend the precipitated extracellular vesicles, centrifuge again, and take the supernatant, which is the crude extracted extracellular vesicles.

[0020] (3) Finally, the crudely extracted extracellular vesicles were transferred into an EPF column, centrifuged, and cell debris and protein aggregates were removed to obtain purified Lactobacillus helveticus BT-B60 extracellular vesicles.

[0021] In a ninth aspect, the present invention provides the application of the aforementioned extracellular vesicles in the preparation of skin antioxidant products.

[0022] Beneficial Effects: Compared with existing technologies, this invention provides a novel *Lactobacillus helveticus* strain BT-B60. This strain exhibits good tolerance to gastric and intestinal fluids, strong intestinal adhesion ability, no hemolytic toxicity, and sensitivity to probiotics and antibiotics, meeting the safety standards for food-grade probiotics. Experimental results show that this strain can promote osteoblast proliferation, inhibit osteoclast formation, increase bone density and bone strength, and improve osteoporosis. It has a clear antioxidant protective effect on liver cells, effectively inhibiting liver oxidative stress damage. Extracellular vesicles prepared using BT-B60 have excellent skin antioxidant function, demonstrating the innovative multi-target and multi-tissue antioxidant characteristics of this strain. In addition, this strain can also inhibit melanin synthesis, synergistically enhancing local skin antioxidant effects and exhibiting multiple benefits in skin whitening. Attached Figure Description

[0023] Figure 1 Gram staining image of strain BT-B60.

[0024] Figure 2 The effect of BT-B60 on lumbar spine bone mineral density in mice.

[0025] Figure 3 To investigate the intervention effect on femoral bone mineral density in mice.

[0026] Figure 4 This refers to the activity of superoxide dismutase (SOD) within vesicles.

[0027] Figure 5 This refers to the glutathione (GSH) content within the vesicles.

[0028] Figure 6 This refers to the malondialdehyde (MDA) content within the vesicles.

[0029] Figure 7 The results show the SOD activity assay in HepG2 cells.

[0030] Figure 8 The results show the detection of GSH content, an antioxidant, in HepG2 cells.

[0031] Figure 9 The results show the detection of antioxidant MDA content in HepG2 cells.

[0032] Figure 10 The effect of BT-B60 on serum ALT and AST levels in mice with CCl4-induced liver injury was investigated. Con was the blank control group, Mod was the model control group, Pos was the positive control group, and BL, BM, and BH were the low, medium, and high dose groups of BT-B60, respectively.

[0033] Figure 11 The effect of BT-B60 on SOD activity in liver tissue of mice with CCl4-induced liver injury.

[0034] Figure 12 The effect of BT-B60 on MDA content in liver tissue of mice with CCl4-induced liver injury.

[0035] Figure 13 The effect of BT-B60 on serum α-MSH activity in mice with CCl4-induced liver injury.

[0036] Figure 14 The effect of BT-B60 on serum TYR activity in mice with CCl4-induced liver injury. Detailed Implementation

[0037] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0038] Example 1: Screening of beneficial human bacterial strains and construction of a strain library

[0039] To obtain strains with both osteoporosis-improving and skin antioxidant functions, a specific beneficial strain library was constructed. Fecal samples from healthy individuals with a BMI of 18.5-24.0 and samples from traditional fermented dairy products (cheese, fermented milk) were collected. After vortexing and serial dilution, the samples were plated onto MRS medium and incubated anaerobically at 37°C for 48 hours. Each gradient was plated three times, and each sample was tested in triplicate. Healthy single colonies were selected, and rapid preliminary identification of the selected colonies was performed using time-of-flight mass spectrometry (MALDI-TOF). Known pathogenic and opportunistic pathogens were discarded. Strains belonging to the probiotic or second-generation probiotic category were inoculated into 5 ml of MRS liquid medium for large-scale culture. After culturing at 35-37℃ for 48 hours, the bacterial count in the fermentation broth was observed by measuring the OD600 and comparing it under a microscope. 2 ml of the strain with a fermentation OD value greater than 0.3 and good growth was added to 2 ml of 30%-40% glycerol solution, pre-cooled, and then frozen at -80℃ for further isolation, purification, and screening.

[0040] Example 2: Screening of strains with free radical scavenging activity

[0041] The dominant bacterial strains screened in Example 1 were cultured in MRS liquid medium at 37°C for 24 hours. The bacterial cells were collected by centrifugation, and the bacterial pellet was washed twice with PBS and resuspended. The bacterial concentration was adjusted to 10. 8 CFU / mL. Add 1 mL of bacterial suspension to 2 mL of 0.2 mmol / L DPPH anhydrous ethanol solution, mix well, and react at room temperature in the dark for 30 min. Centrifuge at 12000 r / min for 2 min, take the supernatant and measure its absorbance at a wavelength of 517 nm. Calculate the DPPH free radical scavenging rate of the probiotic strain using the following formula.

[0042] DPPH clearance rate (%) = [1 - (A sample group - A blank group) / A control group] × 100.

[0043] The difference between the blank group and the sample group was that an equal volume of anhydrous ethanol was used instead of DPPH solution, while the difference between the control group and the sample group was that an equal volume of distilled water was used instead of DPPH solution. The strains with excellent DPPH free radical scavenging effects were obtained through testing, as shown in Table 1.

[0044] Table 1. Scavenging effects of different strains on DPPH free radicals

[0045]

[0046] Example 3: Preservation, Identification and Storage of Strains

[0047] The BT-B60 strain with DPPH free radical scavenging effect screened in Example 2 was preserved, identified, and archived.

[0048] (1) Preservation of strains

[0049] After streaking BT-B60 strain onto MRS agar medium for 48 h, single colonies were selected and placed in 10 ml of MRS liquid medium, and incubated at 37 °C for 16 h. The OD of the culture medium was then measured. 600 When the value is ≥1.2, add glycerol solution to the culture medium at a volume ratio of 1:1. The concentration of the glycerol solution is 30%-40%. Mix well by pipetting and dispensing into 2ml sterile cryovials. Pre-cool at 4℃ for 2h, then pre-freeze at -20℃ for 4h, and finally transfer to a -80℃ freezer or liquid nitrogen for storage.

[0050] (2) Further morphological identification

[0051] Observation of the purified bacterial solution under a microscope shows that the bacteria are short rods, arranged singly, in pairs or in short chains, without spores or flagella; single colonies on MRS agar medium are round, milky white colonies with neat edges, smooth and moist surface, opaque, slightly raised center, and a colony diameter of 0.8-1.2 mm.

[0052] (3) Gram staining

[0053] Gram staining result is Gram positive, such as Figure 1 As shown.

[0054] (4) Molecular biological identification of 16S rRNA

[0055] The 16S rDNA gene sequence of strain TRT-2A was amplified and sequenced using published universal 16S primers (primer sequences: 8F: 5'-AGAFTTTGATCCTGGCTCA-3'; 1510R: 5'-GGTTACCTTGTTACGACTT-3'). The nucleotide sequence of the 16S rDNA of strain BT-B60 is sequence 1 in the sequence listing. After 16S rDNA gene alignment, the similarity rate with Lactobacillus helveticus in Genebank reached 99%. Combined with microbial systematic identification, strain BT-B60 was identified as a subspecies of Bifidobacterium animalis and named Lactobacillus helveticus BT-B60, whose 16S rDNA is shown in SEQ ID NO.1.

[0056] The biologically identified animal subspecies of Bifidobacterium animalis BT-B60 (Lactobacillus helveticus BT-B60) was deposited at the China Center for Type Culture Collection (CCTCC) on November 20, 2025, with accession number CCTCC M 20252624, at Wuhan University, Wuhan, Hubei Province, People's Republic of China.

[0057] SEQ ID NO.1

[0058]

[0059] Example 4: In vitro tolerance test of Lactobacillus helveticus BT-B60

[0060] (1) Artificial gastric juice tolerance test

[0061] The BT-B60 strain was prepared into 1×10⁻⁶ samples. 8 A bacterial suspension of CFU / mL was prepared. 0.5 mL of the bacterial suspension was mixed with 4.5 mL of artificial gastric fluid (pH=2.0, containing 0.3% pepsin), and incubated statically at 37℃. Samples were taken at 2 h and 4 h, serially diluted, and plated on MRS plates. The plates were then anaerobically incubated at 37℃ for 24 h, and the number of colonies was counted. The survival rate was calculated based on the untreated bacterial suspension. Lactobacillus helveticus ATCC 15009 was selected as a control and the experiment was conducted under the same conditions.

[0062] (2) Artificial intestinal fluid tolerance test

[0063] Take 0.5 mL of the above bacterial suspension and mix it with 4.5 mL of artificial intestinal fluid (pH=8.0, containing 0.1% trypsin). Incubate at 37℃ and take samples at 2 h and 4 h respectively. Calculate the survival rate. The results are shown in Table 1.

[0064] The results showed that the BT-B60 strain had better tolerance to artificial gastric and intestinal fluids than the ATCC 15009 strain.

[0065] Table 2. Results of resistance of Lactobacillus helveticus BT-B60 to gastric and intestinal fluids.

[0066]

[0067] Example 5: Intestinal cell adhesion assay of Lactobacillus helveticus BT-B60

[0068] Human colonic epithelial cells (Caco-2) were seeded into 24-well plates and cultured until monolayer confluence. The cells were washed twice with serum-free, antibiotic-free DMEM medium. BT-B60 strain activated to the logarithmic growth phase and Lactobacillus helveticus ATCC15009 were washed twice with sterile PBS, resuspended, and the bacterial concentration was adjusted to 1×10⁻⁶. 9 Add 1 mL of bacterial suspension to each well (CFU / mL, concentration confirmed by plate counting method), incubate at 37°C and 5% CO2 for 4 h, and gently wash three times with sterile phosphate-buffered saline (PBS) to remove unadhered bacteria. Add 0.5 mL of Triton X-100 (0.1%) to each well, lyse cells at room temperature for 10 min, vortex, serially dilute, plate on MRS plates, and anaerobically incubate at 37°C for 24 h. Count the colonies and calculate the adhesion rate.

[0069] Adhesion rate (%) = (Number of adhering colonies ÷ Total number of added colonies) × 100%.

[0070] The results showed that the adhesion rate of BT-B60 strain to Caco-2 cells reached 65.2%, while the adhesion rate of ATCC 15009 under the same conditions was 15.2%. The adhesion rate of BT-B60 strain was significantly higher than that of ATCC 15009 strain, indicating that it has extremely strong intestinal adhesion ability.

[0071] Example 6 Safety test of Lactobacillus helveticus BT-B60

[0072] (1) Hemolysis test

[0073] The BT-B60 strain was inoculated into 5 mL of MRS medium at a 2% inoculum and activated by anaerobic incubation at 37°C for 12 h. Enterococci (β-hemolysis) were used as a positive control, and blank MRS medium as a negative control. 2.5 μL of the activated bacterial solution was taken from each culture and inoculated onto Columbia blood agar plates, anaerobically incubated at 37°C for 48 h, and hemolysis was observed. The results showed that a clear hemolytic zone appeared around the colonies of the positive control, while no hemolytic zone appeared around the colonies of the BT-B60 strain, indicating γ-hemolysis. This suggests that the strain has no hemolytic toxicity and meets the safety requirements for probiotics.

[0074] (2) Drug sensitivity test

[0075] The disk diffusion method (KB method) was used, following the CLSI M45 standard: BT-B60 strain was activated to the logarithmic growth phase, and the bacterial concentration was adjusted to 0.5 McFarland turbidity with sterile physiological saline. The culture was evenly spread on MH agar plates, and antibiotic susceptibility discs for 10 antibiotics (ampicillin, penicillin, erythromycin, clindamycin, tetracycline, chloramphenicol, ciprofloxacin, levofloxacin, sulfamethoxazole, and trimethoprim) were affixed to each plate. The plates were incubated aerobically at 37 °C for 16–18 h, and the diameter of the inhibition zone was measured to determine the strain's susceptibility. Results showed that this strain was sensitive to commonly used probiotic antibiotics such as ampicillin, penicillin, and erythromycin, but resistant to quinolones and sulfonamides. Its resistance to quinolones and sulfonamides was intrinsic to the Lactobacillus genus, with no acquired resistance genes and no risk of horizontal transfer of resistance genes, meeting the safety standards for food-grade probiotics.

[0076] Example 7: Preparation of high-yield industrial freeze-dried powder of Lactobacillus helveticus BT-B60

[0077] To achieve high-density, low-cost industrial-scale fermentation of the BT-B60 strain, this invention optimizes the fermentation medium and process, with the specific steps as follows:

[0078] (1) Culture medium preparation

[0079] Scheme 1 has the following formula (g / L): 12g soy peptone, 8g yeast extract, 6g hydrolyzed whey protein, 25g anhydrous glucose, 6g xylooligosaccharides, 2g calcium carbonate, 2g dipotassium hydrogen phosphate, 1.5g potassium dihydrogen phosphate, 6g sodium acetate, 0.6g magnesium sulfate, 0.5g manganese sulfate, 1.2g L-cysteine ​​hydrochloride, 1mL Tween 80, pH adjusted to 6.8. All raw materials are food-grade.

[0080] Scheme 2 has the following formula (g / L): 10g soybean peptone, 3g yeast extract, 15g anhydrous glucose, 2g xylooligosaccharide, 2g calcium carbonate, 2g dipotassium hydrogen phosphate, 1.5g potassium dihydrogen phosphate, 6g sodium acetate, 0.6g magnesium sulfate, 0.5g manganese sulfate, 1.2g L-cysteine ​​hydrochloride, 1mL Tween 80, and pH adjusted to 6.8. All raw materials are food-grade.

[0081] Option 3 has the following formula (g / L): 18g soy peptone, 15g yeast extract, 10g hydrolyzed whey protein, 30g anhydrous glucose, 2g calcium carbonate, 2g dipotassium hydrogen phosphate, 1.5g potassium dihydrogen phosphate, 6g sodium acetate, 0.6g magnesium sulfate, 0.5g manganese sulfate, 1.2g L-cysteine ​​hydrochloride, 1mL Tween 80, and pH adjusted to 6.8. All ingredients are food-grade.

[0082] (2) Fermentation culture

[0083] For liquid fermentation, the optimized culture medium was sterilized at 115°C for 30 min, cooled to 38°C, and then inoculated at a 10% inoculum to activate the culture to the logarithmic growth phase at a concentration of 1×10⁻⁶. 9 BT-B60 seed culture at CFU / mL was placed in a 50L fermenter for anaerobic fermentation at 37℃, pH maintained at 6.5-6.8, and nitrogen was purged throughout the process to maintain a pressure of 0.05MPa inside the fermenter. The mixture was stirred slowly (50 r / min) for 14 hours, after which the viable cell count in the fermentation broth reached 3.8 × 10⁻⁶. 9 The CFU / mL viable cell count in the fermentation broth has reached the requirements for high-density industrial fermentation production and can meet the needs of large-scale production. The viable cell count in the fermentation broth has remained stable at 2.5 × 10⁻⁶. 9 For samples with CFU / mL or higher, three culture medium regimens were compared. Fermentation validation showed that Regimen 1 had the highest viable cell count (3.8 × 10⁻⁶). 9 The concentration of CFU / mL was significantly higher than that of Scheme 2 (2.5 × 10⁻⁶ CFU / mL). 9 CFU / mL), Scheme 3 (3.2×10) 9 (CFU / mL), therefore, Scheme 1 was determined to be the preferred fermentation medium.

[0084] (3) Centrifugation and freeze drying

[0085] After 12 hours of cultivation, the OD value of the fermentation broth was measured at 600 nm using a UV spectrophotometer to plot the growth curve. Once the plateau phase was confirmed, the fermentation broth was rapidly cooled to below 12°C and centrifuged at 6000 rpm for 20 min using a tubular centrifuge to collect the bacterial sludge. The bacterial sludge was diluted to a water content of 70%, and an equal volume of a special bacterial sludge protectant (composition: water 68%, skim milk powder 12%, mannitol 6%, sucrose 5%, Tween-80 1%, betaine 4%, glutamine 2%, soluble starch 2%) was added. The mixture was thoroughly emulsified with the bacterial sludge freeze-drying protectant and then placed in a freeze dryer for vacuum freeze-drying. After freeze-drying, the moisture content of the material was <4%, and the water activity was 0.05-0.15 aw.

[0086] The freeze-dried BT-B60 bacterial powder has a viable count of 2.2 × 10⁻⁶. 11 With a CFU / g content, it meets the conditions for large-scale industrial fermentation production and can be used as a food additive for lactic acid bacteria powder or for the preparation of microecological intervention preparations.

[0087] Example 8: In vitro evaluation of the effect of Lactobacillus helveticus BT-B60 on improving osteoporosis function.

[0088] (1) Osteoblast proliferation promotion experiment

[0089] After activation, *Lactobacillus helveticus* BT-B60 strain was anaerobically cultured at 37℃ for 24 h, centrifuged at 8000 rpm for 10 min, and the supernatant was sterilized through a 0.22 μm filter membrane. The sterilized supernatant was then added to mouse osteoblast MC3T3-E1 culture medium at 20% (w / w). A blank control group (culture medium only), a negative control group (cells treated with culture medium only), and a positive control group (10...) were established. -8 Cells were treated with vitamin D3 (M) and cultured, and the cell proliferation rate was detected by CCK-8 assay.

[0090] Cell proliferation rate (%) = [(experimental group OD)] 450 - Blank Group OD 450 ) ÷ (OD of negative control group) 450 - Blank Group OD 450 )]×100%.

[0091] The results showed that BT-B60 supernatant promoted the proliferation of mouse osteoblast MC3T3-E1 cells by 32.3%, which was significantly higher than that of the negative control group, indicating that the BT-B60 strain can effectively promote osteoblast proliferation.

[0092] (2) Osteoclast formation inhibition experiment

[0093] Mouse bone marrow macrophages RAW264.7 were used to induce osteoclast formation with RANKL, and bacterial supernatant was added at the same time. The negative control group was induced with RANKL only, without the addition of bacterial supernatant. After culture, TRAP staining and counting were performed to calculate the inhibition rate.

[0094] Osteoclast inhibition rate (%) = [1 - (number of positive cells in the experimental group ÷ number of positive cells in the negative control group)] × 100%.

[0095] The results showed that BT-B60 inhibited osteoclast formation by 65.8%, and the strain significantly inhibited osteoclast activation and bone resorption.

[0096] The ability of Lactobacillus helveticus BT-B60 to promote osteoblast proliferation and inhibit osteoclast formation provides a theoretical basis for subsequent animal experiments and applications.

[0097] Example 9: The ameliorative effect of Lactobacillus helveticus BT-B60 on osteoporosis model mice.

[0098] Twenty-four SPF-grade female mice were selected to establish an ovariectomized osteoporosis model. They were randomly divided into a blank control group, a model control group, a positive control group (alendronate sodium by gavage), and a BT-B60 intervention group (BT-B60 bacterial suspension by gavage). After 4 weeks of post-operative feeding, bone mineral density in the model group was measured. Once significantly lower than that in the blank control group (P<0.01), gavage intervention was initiated for 8 weeks. The gavage volume was 0.2 mL / mouse / day, once daily for 8 consecutive weeks. The experimental animals were housed in an SPF-grade animal facility at a temperature of 22±2℃, relative humidity of 50±5%, and a 12h light / 12h dark cycle, with free access to food and water. Data were analyzed using SPSS 26.0 software using one-way ANOVA. P<0.05 was considered statistically significant.

[0099] The results are as follows Figure 2 and Figure 3 As shown, the BT-B60 intervention group mice showed increased bone mineral density in the lumbar spine and femur, indicating that Lactobacillus helveticus BT-B60 can effectively regulate bone metabolism, increase bone mineral density and bone strength, and has a significant effect on improving osteoporosis.

[0100] Example 10: Antioxidant protective effect of Lactobacillus helveticus BT-B60 extracellular vesicles on skin cells

[0101] The experimental sample was an extracellular vesicle solution of *Lactobacillus helveticus* BT-B60. The preparation method was as follows: *Lactobacillus helveticus* BT-B60 fermentation broth cultured in liquid MRS medium to the stable growth phase (37℃ for 36 h) was centrifuged at 4℃ for 10 min at 4500×g to obtain the metabolic supernatant and *Lactobacillus helveticus* BT-B60 cells. The metabolic supernatant was then filtered through a 0.45 μm filter to further remove cell debris. Extracellular vesicles were extracted from the filtrate using an exosome purification kit: the filtrate was transferred to a new centrifuge tube, ESC (Exosome Concentration Solution) was added, and the mixture was vortexed for 10 s. The mixture was then incubated at 4℃ for 20 h, followed by centrifugation at 10000×g at 4℃ for 1 h to precipitate the extracellular vesicles. The precipitated extracellular vesicles were resuspended in pre-chilled PBS at 4℃, centrifuged at 12000×g at 4℃ for 2 min, and the supernatant was collected as the crude extracted extracellular vesicles. Finally, the crudely extracted extracellular vesicles were transferred to an EPF (Exosome Purification Filter) column and centrifuged at 4 ℃ and 3000×g for 10 min to further remove cell debris and protein aggregates, finally obtaining purified Lactobacillus helveticus BT-B60 extracellular vesicles (BT-B60 Baevs). The prepared extracellular vesicles were diluted to 1.0×10⁻⁶ with physiological saline. 8 Extracellular vesicle solution of *Lactobacillus helveticus* BT-B60 was obtained by measuring particles / mL. Human skin fibroblast HSF was seeded into 96-well plates and cultured to 80% confluence. Cells were then divided into a blank control group (BK), an oxidative damage group (Ctrl), a BT-B60 Baevs intervention group (BT-B60 Baevs), and a vitamin C control group (VC), and cultured for 24 h. The activities of superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) within the vesicles were measured. Results are shown below. Figure 4-6 As shown, the BT-B60-Baevs intervention group showed increased intracellular SOD activity, increased GSH content, and decreased MDA content, comparable to the vitamin C control group. This indicates that the fermentation products of the BT-B60 strain can significantly scavenge free radicals in skin cells and have excellent skin antioxidant function.

[0102] Example 11: Verification of the antioxidant function of Lactobacillus helveticus BT-B60 in liver cells

[0103] This embodiment uses an in vitro human hepatocellular carcinoma HepG2 cell oxidative damage model to verify the antioxidant protective ability of *Lactobacillus helveticus* BT-B60 on liver cells. The liver cell antioxidant evaluation system constructed in this experiment differs from the aforementioned skin cell antioxidant system, clarifying the strain's specific antioxidant activity against liver tissue at the in vitro level, laying the foundation for subsequent in vivo animal experiments, and demonstrating the innovative characteristics of the strain's multi-target and multi-tissue antioxidant activity.

[0104] Human hepatocellular carcinoma HepG2 cells were seeded into 96-well cell culture plates and cultured until the cells adhered and confluent to the plate at 80%. The cells were then randomly divided into a blank control group, an oxidative damage group, a positive control group (50 μmol / L silymarin, a classic hepatoprotective drug with the effects of scavenging free radicals, resisting oxidative damage, and protecting hepatocytes, which served as the positive control in this experiment), a BT-B60 intervention group, and a Lactobacillus helveticus ATCC 15009 control group.

[0105] An oxidative damage model was established using 0.2 mmol / L H2O2. After treatment, each group was cultured for 48 h, and the SOD / GSH / MDA levels were measured. The results are as follows: Figures 7-9 As shown, the BT-B60 intervention group showed upregulated SOD and GSH activities and decreased MDA content, indicating that it has a clear antioxidant protective effect on liver cells. Moreover, this effect is tissue-specific and independent of the skin cell antioxidant system verified in the previous invention, demonstrating the innovative multi-target and multi-tissue antioxidant characteristics of this strain.

[0106] Example 12: Antioxidant protective effect of Lactobacillus helveticus BT-B60 on a mouse model of CCl4-induced liver oxidative damage.

[0107] Sixty SPF-grade male C57BL / 6 mice were randomly divided into 6 groups of 10 mice each: a blank control group, a model control group, a positive control group (100 mg / kg silymarin), and low / medium / high dose BT-B60 groups (1×10⁻⁶). 9 5×10 9 1×10 10 (CFU / mL bacterial suspension). Except for the blank control group, all other groups established a liver oxidative damage model by intraperitoneal injection of 10% CCl4-ol olive oil solution, followed by continuous gavage intervention for 8 weeks. After the last administration, serum, liver tissue and back skin tissue samples were collected for subsequent multi-indicator detection.

[0108] Serum ALT and AST activities, serum and liver tissue SOD and GSH-Px activities, and MDA content were measured, and liver tissue was subjected to HE staining pathological analysis.

[0109] The results are as follows Figure 10-12 As shown, compared with the model control group, each dose group of BT-B60 significantly reduced the activity of ALT, AST and MDA in mouse serum, increased the activity of SOD in liver tissue, and improved the pathological damage such as CCl4-induced hepatocyte swelling and fatty degeneration. This indicates that the BT-B60 strain has excellent in vivo liver antioxidant protection and can effectively inhibit liver oxidative stress damage.

[0110] Example 13: The effect of Lactobacillus helveticus BT-B60 in inhibiting melanin synthesis by regulating liver metabolism.

[0111] The mice used in this example were the same batch of CCl4-induced liver oxidative damage model mice as in Example 12. Based on serum and back skin tissue samples collected after the last administration, the regulatory effect of BT-B60 on melanin synthesis was evaluated. Serum α-MSH and TYR were detected using an ELISA kit; skin SOD and MDA were detected using a colorimetric method; and skin melanin content was determined after extraction using the sodium hydroxide lysis method.

[0112] The activities of α-MSH and TYR in the serum of mice in each group, the SOD activity in the liver and skin tissues, and the melanin content in the skin tissue were measured. The results are as follows: Figure 13 and 14 As shown.

[0113] The results showed that, compared with the model control group, BT-B60 downregulated serum α-MSH and TYR activities, indicating that BT-B60 can inhibit melanin synthesis from the root by improving liver oxidative stress, effectively reduce melanin deposition in liver and skin tissues, and synergize with local skin antioxidant effects, thus having multiple effects in whitening.

[0114] The embodiments of the present invention have been described in detail above with reference to specific examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A strain of *Lactobacillus helveticus* with osteoporosis-improving function, characterized in that, The Lactobacillus helveticus mentioned is Lactobacillus helveticus BT-B60, deposited at the China Center for Type Culture Collection (CCTCC) on November 20, 2025, with accession number CCTCC NO: M 20252624.

2. A composition, characterized in that, The composition comprises Lactobacillus helveticus BT-B60 as described in claim 1.

3. The composition according to claim 2, characterized in that, The composition is a lyophilized powder containing *Lactobacillus helveticus* BT-B60 as described in claim 1; preferably, the activity of the strain in the lyophilized powder is 1 × 10⁻⁶. 10 ~1×10 12 CFU / g.

4. The fermentation medium for Lactobacillus helveticus BT-B60 according to claim 1, characterized in that, The fermentation medium comprises the following components: Soybean peptone 10-14 g / L, yeast extract 6-10 g / L, hydrolyzed whey protein 4-8 g / L, anhydrous glucose 20-30 g / L, xylooligosaccharides 4-8 g / L, calcium carbonate 1-3 g / L, dipotassium hydrogen phosphate 1-3 g / L, potassium dihydrogen phosphate 0.5-2.5 g / L, sodium acetate 4-8 g / L, magnesium sulfate 0.2-1.0 g / L, manganese sulfate 0.1-1.0 g / L, L-cysteine ​​hydrochloride 0.8-1.4 g / L, Tween 80 0.5-1.5 mL / L, pH adjusted to 6-7.

5. The use of Lactobacillus helveticus BT-B60 of claim 1, or the composition of claim 2 or 3, in the preparation of products for improving osteoporosis.

6. The use of Lactobacillus helveticus BT-B60 of claim 1, or the composition of claim 2 or 3, in the preparation of products that inhibit oxidative damage to the liver.

7. The use of Lactobacillus helveticus BT-B60 of claim 1, or the composition of claim 2 or 3, in the preparation of products that inhibit melanin synthesis.

8. An extracellular vesicle, characterized in that, The extracellular vesicles were prepared using Lactobacillus helveticus BT-B60 as described in claim 1.

9. The method for preparing extracellular vesicles according to claim 8, characterized in that, Includes the following steps: (1) Centrifuge the fermentation broth of Lactobacillus helveticus BT-B60 cultured to the stable growth phase to obtain metabolic supernatant and Lactobacillus helveticus BT-B60 cells. Filter the metabolic supernatant to further remove cell debris. (2) Add exosome extraction reagent to the filtrate obtained in step (1), mix well, let stand, then centrifuge to precipitate extracellular vesicles, resuspend the precipitated extracellular vesicles, centrifuge again, and take the supernatant, which is the crude extracted extracellular vesicles. (3) Finally, the crudely extracted extracellular vesicles were transferred into an EPF column, centrifuged, and cell debris and protein aggregates were removed to obtain purified Lactobacillus helveticus BT-B60 extracellular vesicles.

10. The use of the extracellular vesicles according to claim 8 in the preparation of skin antioxidant products.

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